Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins

Autofluorescence imaging (AFI) has greatly accelerated in the last decade, way past its origins in detecting endogenous signals in biological tissues to identify differences between samples. There are many endogenous fluorescence sources of contrast but the most robust and widely utilized have been those associated with metabolism. The intrinsically fluorescent metabolic cofactors Nicotinamide adenine dinucleotide (NAD+/NADH) and Flavin adenine dinucleotide (FAD/FADH2) have been utilized in a number of AFI applications including basic research, clinical and pharmaceutical studies. Fluorescence lifetime imaging microscopy (FLIM) has emerged as one of the more powerful AFI tools for NADH and FAD characterization due to its unique ability to non-invasively detect metabolite bound and free states and quantitate cellular redox ratio. However, despite this widespread biological use, many standardization methods are still needed to extend FLIM based AFI into a fully robust research and clinical diagnostic tools. FLIM is sensitive to a wide range of factors in the fluorophore microenvironment and there a number of analysis variables as well. To this end there has been an emphasis on developing imaging standards and ways to make the image acquisition and analysis more consistent. However biological conditions during FLIM based AFI imaging are rarely considered as key sources of FLIM variability. Here we present several experimental factors with supporting data of the cellular microenvironment such as confluency, pH, inter/intra cellular heterogeneity, and choice of cell line that need to be considered for accurate quantitative FLIM based AFI measurement of cellular metabolism.

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“…Imaging was carried out on a custom-built multiphoton microscope that was previously described (46–48). Refer Figure 3 for the microscope scheme.…”

Section: Methodsmentioning

confidence: 99%

“…Imaging was carried out on a custom-built multiphoton microscope that was previously described (46)(47)(48) for the microscope scheme). Autofluorescence lifetime imaging was excited using a mode-locked tunable (690-1040 nm) ultrafast laser working at 80 MHz (Mai Tai DeepSee; Spectra Physics, Santa Clara, CA).…”

Section: Imagingmentioning

confidence: 99%

Autofluorescence lifetime imaging of cellular metabolism: Sensitivity toward cell density, pH, intracellular, and intercellular heterogeneity

Chacko

Eliceiri

2018

Cytometry Pt A

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“…FLIM has also been performed in plants such as Arabidopsis, where FLIM estimated vacuolar pH inside intact plant cells with the lifetime of anthocyanin. 267…”

Section: In Vivo Autofluorescence Flimmentioning

confidence: 99%

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

et al. 2020

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Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy. Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications. Approach: This review covers FLIM principles and theory, including advantages over intensitybased fluorescence measurements. Fundamentals of FLIM instrumentation in time-and frequencydomains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell-and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed. Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

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“…However, fluorescence lifetime imaging (FLIM) which is often used to study dynamic quenching which reduces the fluorescence lifetime of the quenched molecule [44] might not prove useful as fluorescent lifetimes are not typically reduced by static quenching [34]. FLIM has already been applied to anthocyanin, with Arabidopsis anthocyanin showing a pH-dependent lifetime of 0.1 to 0.5 nanoseconds [45], and it might be that a lack of a change in carboxyfluorescein lifetime in the presence of anthocyanin would confirm that static quenching. In vivo quenching of GFP by anthocyanin might also be investigated.…”

Section: Future Directionsmentioning

confidence: 99%

“…Instead, the non-trivial exercise of generating stable transformants of onion [46] or garlic [47] lines exhibiting epidermal anthocyanin, and expressing a vacuolar-targeted GFP might need to be used. However, stably transformed Arabidopsis lines expressing vacuolar targeted GFP might be induced to form anthocyanins through high sucrose treatments and other abiotic stresses [45,48,49].…”

Section: Future Directionsmentioning

confidence: 99%

Anthocyanin in the Vacuole of Red Onion Epidermal Cells Quenches Other Fluorescent Molecules

Collings

2019

Plants

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Peels from the inner epidermis of onion bulbs are a model system in plant cell biology. While the inner epidermis of red onions is characteristically white, small patches of cells sometimes redden, containing vacuolar anthocyanin. This study investigated the spectroscopic properties of these anthocyanic cells. When fluorescent dyes were loaded into the vacuole of onion epidermal cells, the anthocyanic cells showed decreased dye fluorescence. This decrease was observed for fluorescein and carboxyfluorescein that are pumped into the vacuole by anion transporters, for acridine orange which acid loads into the vacuole, and for the fluorescent sugar analogue esculin loaded into the vacuole by sucrose transporters. Similar decreases in carboxyfluorescein fluorescence were observed when dye was loaded into the vacuoles of several other plant species, but decreases were not observed for dyes resident in the tonoplast membrane. As cellular physiology was unaffected in the anthocyanic cells, with cytoplasmic streaming, vacuolar and cytoplasmic pH not being altered, the decreased dye fluorescence from the anthocyanic cells can be attributed to fluorescence quenching. Furthermore, because quenching decreased with increasing temperature. It was concluded, therefore, that vacuolar anthocyanin can statically quench other fluorescent molecules in vivo, an effect previously demonstrated for anthocyanin in vitro.2 of 15 pH-dependant, which means that anthocyanin colour, and the colour of the plant, will depend upon vacuolar pH. Anthocyanin colour may also vary because anthocyanins can interact with themselves, stacking to form super-molecular structures, and can interact with other flavonoid pigments, stabilising the coloured forms at acidic pHs, and also interact with metal ions [1][2][3][4].Like many flavonoids, anthocyanins can be weakly autofluorescent with in vitro excitation and emission peaks in the UV [4,5]. However, in vivo excitation of anthocyanins in both Arabidopsis thaliana and onion (Allium cepa) results in red fluorescence, and has allowed for direct visualisations of vacuole structure and dynamics [6][7][8].The flavonoid biosynthetic pathway, a complex series of reactions in which coumaroyl-CoA is sequentially modified to form various end-products, results in the formation of anthocyanins [9]. The enzymes controlling this pathway vary slightly between species resulting in the variability in the anthocyanidin core and attached sugars. Following their synthesis in the cytoplasm, the vacuolar transport of anthocyanins occurs through several pathways. These include ER-derived vesicles [6,10] and a tonoplast-bound glutathione S-transferase-like transporter that sequesters various glutathione conjugates and anthocyanins into the vacuole [11,12]. The mechanism(s) underlying this second process are unclear as anthocyanin-glutathione conjugates remain undiscovered [12]. The multi-drug resistance-like protein is another membrane-bound transporter known to transport anthocyanin into maize vacuoles [1].It is thought that anth...

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Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins

Cited by 23 publications

References 57 publications

Autofluorescence lifetime imaging of cellular metabolism: Sensitivity toward cell density, pH, intracellular, and intercellular heterogeneity

Autofluorescence lifetime imaging of cellular metabolism: Sensitivity toward cell density, pH, intracellular, and intercellular heterogeneity

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

Anthocyanin in the Vacuole of Red Onion Epidermal Cells Quenches Other Fluorescent Molecules

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